Gene participating in acetic acid tolerance, acetic acid bacteria bred using the gene, and process for producing vinegar with the use of the acetic acid bacterium

ABSTRACT

The present invention is intended to provide novel genes participating in acetic acid tolerance of acetic acid bacteria, and a method of improving acetic acid tolerance of microorganisms, particularly that of acetic acid bacteria by using the genes, further a method of efficiently producing vinegar with acetic acid at higher concentration by using acetic acid bacteria whose acetic acid tolerance is improved. In the present invention, novel genes having a function for improving acetic acid tolerance on practical level were cloned from practical acetic acid bacteria belonging to the genus  Gluconacetobacter  by a method of obtaining genes from chromosomal DNA library that enable to grow on the medium at a high concentration of acetic acid. Further, in transformants in which the genes were introduced into acetic acid bacteria, acetic acid tolerance was remarkably increased, and when the transformants are subjected to aeration culture in the presence of ethanol, the growth lag-time can be shortened, and the growth rate can also be improved, moreover the final acetic acid concentration can be remarkably improved.

TECHNICAL FIELD TO WHICH THE INVENTION PERTAINS

The present invention relates to genes that encode protein having afunction of enhancing acetic acid tolerance derived from microorganisms,microorganisms whose copy numbers of the genes are amplified,particularly acetic acid bacteria which belong to the genus Acetobacteror the genus Gluconacetobacter, and a method of efficiently producingvinegar containing acetic acid at a high concentration by using thesemicroorganisms.

PRIOR ART

Acetic acid bacteria are microorganisms widely used in producingvinegar, and particularly acetic acid bacteria which belong to the genusAcetobacter or the genus Gluconacetobacter are utilized for industrialacetic acid fermentation.

In acetic acid fermentation, ethanol in medium is oxidized to aceticacid by acetic acid bacteria, and acetic acid resultantly accumulates inthe medium, but acetic acid is inhibitive for acetic acid bacteria aswell, and growth ability and fermentation ability of acetic acidbacteria gradually decrease as the concentration of acetic acid in themedium increases.

Accordingly, it is required in acetic acid fermentation that growthability and fermentation ability do not decrease even at higherconcentration of acetic acid, i.e. development of acetic acid bacteriahaving strong acetic acid tolerance is required. It has been attemptedas one of the means that genes participating in acetic acid tolerance(acetic acid resistance-genes) are cloned and acetic acid bacteria arebred and improved by using the acetic acid resistance-genes.

As for findings relating to acetic acid resistance-genes of acetic acidbacteria heretofore, three genes (aarA, aarB, and aarC) clustering havebeen cloned as complementary genes which can restore the acetic acidsensitive mutants of acetic acid bacteria belonging to the genusAcetobacter, to the original tolerance (see non-patent literature 1, forinstance).

Among them, it was presumed that aarA gene encodes citric acid synthaseand aarC gene encodes enzyme relating to assimilation of acetic acid,while the function of aarB gene has been uncertain (see non-patentliterature 2, for instance).

In the transformants obtained by transformation with cloned genefragment containing these three acetic acid resistance-genes onmulti-copy plasmid to Acetobacter aceti subspecies xylinum IF03288strain, the level of improvement of acetic acid tolerance was only low,and it was uncertain if ability of these three acetic acidresistance-genes in actual acetic acid fermentation was improved or not(see non-patent literature 1, for instance).

Meanwhile, there has been a disclosed example in which improvement ofthe final acetic acid concentration in acetic acid fermentation wasconfirmed by introducing gene encoding membrane-bound aldehydedehydrogenase (ALDH) cloned from acetic acid bacteria into acetic acidbacteria (see non-patent literature 2, for instance). However, sinceALDH is not an enzyme directly participating in acetic acid tolerancebut that having a function of oxidizing aldehyde, it could not beconcluded whether gene encoding ALDH was the exact acetic acidresistance-gene.

Patent Literature 1

Japanese Laid-Open Patent Application No. 1991-219878

Patent Literature 2

Japanese Laid-Open Patent Application No. 1990-2364

Patent Literature 3

Japanese Laid-Open Patent Application No. 1985-9489

Patent Literature 4

Japanese Laid-Open Patent Application No. 1985-9488

Non-patent Literature 1

Journal of Bacteriology, vol. 172, 2096-2104, 1990

Non-patent Literature 2

Journal of Fermentation and Bioengineering, vol. 76, 270-275, 1993

Non-patent Literature 3

Applied of Environment and Microbiology, vol. 55, 171-176, 1989

Non-patent Literature 4

Agricultural and Biological Chemistry, vol. 52, p. 3125-3129, 1988

Non-patent Literature 5

Agricultural and Biological Chemistry, vol. 49, p. 2091-2097, 1985

Non-patent Literature 6

Bioscience, Biotechnology and Biochemistry, vol. 58, p. 974-975, 1994

PROBLEM TO BE SOLVED BY THE INVENTION

As mentioned above, no example that has elucidated acetic acid toleranceof acetic acid bacteria on genetic level and has succeeded in thedevelopment of practical acetic acid bacteria having high acetic acidtolerance has been reported heretofore. However, development of aceticacid bacteria superior in acetic acid tolerance would allow theperformance of acetic acid fermentation at higher concentration thanthat conventional one and efficient production of acetic acid at a highconcentration and vinegar at a higher concentration. Therefore, thepresent inventors attempted again to elucidate the improvement of aceticacid tolerance of acetic acid bacteria on genetic level.

As a result of consideration from various aspects, and from the viewpoint that it was important to obtain novel acetic acid resistance-genesencoding proteins that have a function capable of improving acetic acidtolerance on practical level and to breed acetic acid bacteria havingstronger acetic acid tolerance with the use of the obtained acetic acidresistance-genes, the present inventors have newly set novel technicaltasks to furnish novel genes participating in acetic acid tolerancederived from microorganisms belonging to acetic acid bacteria, and tofurnish a method of improving acetic acid tolerance of microorganisms byusing the genes, particularly a method of improving acetic acidtolerance of microorganisms belonging to acetic acid bacteria, further amethod of efficiently producing vinegar having acetic acid at a highconcentration by using the acetic acid bacteria whose acetic acidtolerance was improved.

MEANS FOR SOLVING PROBLEMS

The present inventors hypothesized that specific genes participating inacetic acid tolerance that do not exist in other microorganisms shouldexist in acetic acid bacteria capable of growing and fermenting even inthe presence of acetic acid, and the present inventors obtained a novelconcept that the use of these genes would allow improvement of aceticacid tolerance of microorganisms more than before, further it wouldallow the development of an efficient method of producing novel vinegarcontaining acetic acid at a high concentration which could not beconventionally obtained.

As for the conventional method of obtaining acetic acidresistance-genes, it was popular that genes which complement mutant ofacetic acid bacteria with acetic acid sensitivity were cloned.

However, thinking that it was difficult to find acetic acidresistance-genes which were industrially useful by such a method, thepresent inventors have developed a method as that of finding acetic acidresistance-genes from acetic acid bacteria, in which chromosomal DNAlibrary of acetic acid bacteria was constructed, this chromosomal DNAlibrary was transformed into acetic acid bacteria, that can generallygrow only under up to approximately 1% acetic acid and the genes capableof growing the strain in the presence of 2% acetic acid as well, wereobtained by screening.

According to the use of this method, the present inventors havesucceeded for the first time in cloning novel acetic acidresistance-genes that have a function of improving acetic acid toleranceon practical level from acetic acid bacteria belonging to the genusGluconacetobacter practically used in producing vinegar.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

This is a schematic view showing a restriction enzyme map of a genefragment (pP1) derived from Gluconacetobacter entanii cloned by usingPstI, the location of acetic acid resistance-gene and the insert intopSPT.

FIG. 2

This is a schematic view showing a restriction enzyme map of a genefragment (pP2) derived from Acetobacter aceti cloned by using inversePCR method, the location of acetic acid resistance-gene and an insertinto pSPT2.

FIG. 3

This is a figure showing the time course in cultivation of thetransformant whose copy numbers of acetic acid resistance-gene derivedfrom Gluconacetobacter entanii were amplified.

FIG. 4

This is a figure showing the time course in acetic acid fermentation athigher temperature by the transformant whose copy numbers of acetic acidresistance-gene derived from Gluconacetobacter entanii were amplified.

FIG. 5

This is a figure showing an amino acid sequence (SEQ. ID No. 2) of theprotein deduced by an acetic acid resistance-gene derived fromGluconacetobacter entanii.

FIG. 6

This is a figure showing an amino acid sequence (SEQ. ID No. 4) of theprotein deduced by an acetic acid resistance-gene derived fromAcetobacter aceti.

FIG. 7

This is a figure showing primer 1.

FIG. 8

This is a figure showing primer 2.

FIG. 9

This is a figure showing primer 3.

FIG. 10

This is a figure showing primer 4.

FIG. 11

This is a figure showing primer 5.

FIG. 12

This is a figure showing primer 6.

The obtained acetic acid resistance-genes showed homology with proteinreferred to as serine palmitoyltransferase that catalyze the firstprocess of sphingolipid synthesis found in Sphingomonas and the like,and they were presumed as genes that encode serine palmitoyltransferasesof acetic acid bacteria, as a result of homology search onDDBJ/EMBL/Genbank and SWISS-PROT/PIR.

However, the aforementioned gene of serine palmitoyltransferase fromgenus Sphingomonas was the only example in prokaryotes heretofore.

Further, one of the obtained serine palmitoyltransferase genes of aceticacid bacteria had 46.3% homology on amino acid sequence level with knownserine palmitoyltransferase gene found in the genus Sphingomonas and ithad approximately 25% homology with that of mice, the ratio of whichwere so low that it was confirmed that it was similar to other serinepalmitoyltransferase genes to some extent, but it was novel geneencoding the novel protein (it is sometimes referred to as protein SPT)specific to acetic acid bacteria.

In addition, in the transformants in which the genes were inserted toplasmid vectors and transformed into acetic acid bacteria and their copynumbers were amplified, remarkable improvement in acetic acid tolerancecould be seen. When the transformants were subjected to aerobiccultivation in the presence of ethanol, we found that growth rate andproduction rate were improved as well as growth lag time was shortened,further final acetic acid concentration was remarkably improved as aresult. The present inventors further have succeeded in determination ofthe nucleotide sequence and the deduced amino acid sequence of geneticDNA encoding thereof. According to these findings, we have completed thepresent invention.

DISCLOSURE OF THE INVENTION

The embodiment of the present invention is as follows.

(1) A protein SPT described in following (A) or (B):

(A) A protein having an amino acid sequence shown in SEQ. ID No. 2 inthe sequence listing.

(B) A protein consisting of an amino acid sequence comprisingsubstitution, deletion, insertion, addition, or inversion of one orseveral amino acids in an amino acid sequence shown in SEQ. ID No. 2 inthe sequence listing and having a function of enhancing acetic acidtolerance.

(2) A DNA of a gene encoding the protein SPT described in following (A)or (B):

(A) A protein having an amino acid sequence shown in SEQ. ID No. 2 inthe sequence listing.

(B) A protein consisting of an amino acid sequence comprisingsubstitution, deletion, insertion, addition, or inversion of one orseveral amino acids in an amino acid sequence shown in SEQ. ID No. 2 inthe sequence listing and having a function of enhancing acetic acidtolerance.

(3) The DNA of a gene according to (2), that is a DNA described infollowing (a) or (b):

(a) A DNA that comprises a nucleotide sequence consisting of nucleotides187 to 1386 shown in SEQ. ID No. 1 in the sequence listing within thenucleotide sequence.

(b) A DNA that hybridizes with a probe comprising a nucleotide sequenceconsisting of nucleotides 187 to 1386 shown in SEQ. ID No. 1 in thesequence listing within the nucleotide sequence or a part thereof undera stringent condition, and encodes protein having a function ofenhancing acetic acid tolerance.

(4) A protein SPT2 described in following (A) or (B):

(A) A protein having an amino acid sequence shown in SEQ. ID No. 4 inthe sequence listing.

(B) The protein SPT2 consisting of an amino acid sequence comprisingsubstitution, deletion, insertion, addition, or inversion of one orseveral amino acids in an amino acid sequence shown in SEQ. ID No. 4 inthe sequence listing and having a function of enhancing acetic acidtolerance.

(5) A DNA of a gene encoding the protein SPT2 described in following (A)or (B):

(A) A protein having an amino acid sequence shown in SEQ. ID No. 4 inthe sequence listing.

(B) The protein SPT2 consisting of an amino acid sequence comprisingsubstitution, deletion, insertion, addition, or inversion of one orseveral amino acids in an amino acid sequence shown in SEQ. ID No. 4 inthe sequence listing and having a function of enhancing acetic acidtolerance.

(6) The DNA of a gene according to (5), that is a DNA described infollowing (A) or (B):

(A) A DNA that comprises a nucleotide sequence consisting of nucleotides110 to 1321 shown in SEQ. ID No. 3 in the sequence listing within thenucleotide sequence.

(B) A DNA that hybridizes with a probe generated from a nucleotidesequence consisting of nucleotides 110 to 1321 shown in SEQ. ID No. 3 inthe sequence listing within the nucleotide sequence or a part thereofunder a stringent condition, and encodes protein having a function ofpromoting growth rate.

(7) Microorganisms wherein acetic acid tolerance thereof is enhanced byamplifying an intracellular copy number of the DNA according to any oneof said (2), (3), (5), or (6).

(8) The microorganisms according to said (7), wherein the microorganismsare acetic acid bacteria belonging to the genus Acetobacter or the genusGluconacetobacter.

(9) A method of producing vinegar, wherein microorganisms having alcoholoxidation ability among the microorganisms according to said (7) or (8),are cultured on medium containing alcohol and they produce andaccumulate acetic acid in the medium, and a novel vinegar obtainedtherefrom whose acetic acid content is high (10-17.5%).

(10) A recombinant plasmid pUSPT (FERM BP-7932) including the DNAaccording to said (2) or (3), or a recombinant plasmid pUSPT2 (FERMBP-8304) including the DNA according to said (5) or (6).

(11) A recombinant plasmid comprising a DNA fragment having at least anucleotide sequence shown in SEQ. ID No. 1 or 3, for instance plasmidpSPT or plasmid pSPT2 obtained from inserting these DNA fragments to anacetic acid bacteria-Escherichia colt shuttle vector (a multi-copyvector) pMV24, and/or a transformant which is obtained by introducingthese plasmids pSPT and pSPT2 into Acetobacter aceti No. 1023 (FERMBP-2287) or Acetobacter altoacetigenes MH-24 strain (FERM BP-491).

By the present invention, tolerance against acetic acid can be providedtoward microorganisms. Moreover, in microorganisms having alcoholoxidation ability, particularly in acetic acid bacteria, toleranceagainst acetic acid can be remarkably improved and the ability ofefficiently accumulating acetic acid at a high concentration in themedia can be provided.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in more detail.

(1) The DNA of the Present Invention

The DNA of the present invention comprises homology with serinepalmitoyltransferase of the genus Sphingomonas to some extent, and anucleotide sequence that can encode protein having a function ofimproving acetic acid tolerance and comprising an amino acid sequenceshown in SEQ. ID No. 2 in the sequence listing, and it comprisesregulating elements and the structural gene.

The DNA of the present invention can be obtained from chromosomal DNA ofGluconacetobacter entanii as follows.

First, chromosomal DNA library of Gluconacetobacter entanii, forinstance Acetobacter altoacetigenes MH-24 strain (deposited as FERMBP-491 with International Patent Organism Depositary) is prepared.Meanwhile chromosomal DNA is obtained by the method disclosed in patentliterature 3.

Next, a chromosomal DNA library is prepared in order to isolate aceticacid resistance-genes from the obtained chromosomal DNA. To begin with,the chromosomal DNA is partially digested by suitable restrictionenzymes to obtain various DNA fragment-mixtures. Various kinds ofrestriction enzymes can be used by adjusting cleavage reaction time andthe like to control the extent of cleavage. For instance, chromosomalDNA is digested by the restriction enzyme of Sau3AI at the 30° C. orhigher, preferably 37° C., at enzyme concentration of 1-10 units/ml forvarious time periods (1 min-2 h). Meanwhile, PstI was used inafter-mentioned Examples.

Then digested fragments of chromosomal DNA are ligated to vector DNAthat can replicate autonomously in acetic acid bacteria to preparerecombinant DNA. Specifically, this vector DNA is completely digested bythe action of a restriction enzyme which yields a terminal nucleotidesequence complementary to the PstI restriction enzyme used for digestingof chromosomal DNA, for instance PstI, under the condition at thetemperature of 30° C. and enzyme concentration of 1-100 units/ml for 1 hor longer.

Subsequently, the obtained chromosomal DNA fragment-mixtures in theabove-mentioned manner are mixed with the digested vector DNA, thenrecombinant DNA is obtained by the action of T4DNA ligase to the mixtureunder the condition at the temperature of 4-16° C., at enzymeconcentration of 1-100 units/ml for 1 h or longer, preferably 6-24 h.

Using the obtained recombinant DNA, acetic acid bacteria generallyincapable of growing in the presence of acetic acid at higher than 1%concentration on agar media, for instance Acetobacter aceti No. 1023strain (deposited as FERM BP-2287 with International Patent OrganismDepositary) are transformed, and cultivated on agar media containing 2%acetic acid. DNA fragments including acetic acid resistance-genes can beobtained by cultivating there generated colonies into liquid media, thenrecovering plasmids from the obtained bacterial cells.

As for the DNA of the present invention, DNA comprising a nucleotidesequence shown in SEQ. ID No. 1 or 3 in the sequence listing isspecifically exemplified, among which the nucleotide sequence consistingof nucleotides 187 to 1386 shown in SEQ. ID No. 1 within the nucleotidesequence or nucleotides 110 to 1321 shown in SEQ. ID No. 3 within thenucleotide sequence is coding region.

As the result of homology search on DDBJ/EMBL/Genbank and SWISS-PROT/PIRabout the nucleotide sequence shown in SEQ. ID No. 1, an amino acidsequence shown in SEQ. ID No. 2 (FIG. 3: corresponding to nucleotides187 to 1386), the nucleotide sequence shown in SEQ. ID No. 3 or an aminoacid sequence shown in SEQ. ID No. 4 (FIG. 4: corresponding tonucleotides 110 to 1321), the nucleotide sequence shown in SEQ. ID No. 1or the amino acid sequence shown in SEQ. ID No. 2 showed 46.3% homologywith SPT1 gene of Sphingomonas paucimobilis, while showed 26.3% and24.8% homologies with LCB1 gene and LCB2 gene of mice, respectively onamino acid sequence level, and the genes were presumed as those encodingserine palmitoyltransferases, but each of their homologies was as low as50% or less, so that it was apparent that this was novel and differentfrom these genes.

Further, the nucleotide sequence shown in SEQ. ID No. 2 or the aminoacid sequence shown in SEQ. ID No. 4 showed 46.7% homology with SPT1gene, while showed 22.6% and 19.8% homologies with LCB1 gene and LCB2gene of mice, respectively on amino acid sequence level, and the geneswere presumed as those of encoding serine palmitoyltransferases, buteach of their homologies was as low as 50% or less and it was apparentthat this was novel and different from these genes.

Meanwhile, it has been unknown at all that aforementioned SPT gene andthe like participate in acetic acid tolerance.

Further, the DNA of the present invention was identified that it is anovel gene having a function of enhancing the acetic acid tolerancewhich is different from previously obtained acetic acid resistance-genes(aarA, aarB and aarC) of acetic acid bacteria, ADH gene having afunction of enhancing acetic acid tolerance or the like.

As the nucleotide thereof was revealed, for instance the DNA of thepresent invention can also be obtained by polymerase chain reaction (PCRreaction) using genomic DNA of the acetic acid bacteriaGluconacetobacter entanii as a template and oligonucleotide synthesizedbased on the nucleotide sequence as a primer, or by hybridization usingoligonucleotide synthesized based on the nucleotide sequence as a probe,as well.

As for the synthesis of the oligonucleotide, it can be synthesized, forinstance using commercially available various DNA synthesizers in theconventional manner. In addition, PCR reaction can be performed in theconventional manner using Thermal Cycler Gene Amp PCR System 2400(Applied Biosystems) and Taq DNA polymerase (Takara Shuzo Co., LTD.),KOD-Plus—(TOYOBO Co., LTD.) and the like.

As for the DNA encoding the protein having a function of enhancingacetic acid tolerance of the present invention, it may be a DNA encodingprotein where one or several amino acid is deleted, substituted,inserted or added at one or several sites, as long as the function ofenhancing acetic acid tolerance of the encoded protein is not impaired.

The DNA encoding a protein substantially identical to such proteinhaving a function of enhancing acetic acid tolerance, can also beobtained by modifying the nucleotide sequence so that amino acid at aparticular site is deleted, substituted, inserted or added bysite-directed mutagenesis, for instance. Further, the modified DNA suchas aforementioned one can also be obtained by conventionally knownmutagenesis treatments.

In addition, as an amino acid sequence of protein and a nucleotidesequence encoding thereof are generally known that they are slightlydifferent among species, strains, mutants, and variants, the DNAencoding substantially identical proteins can be obtained from generalacetic acid bacteria, among which, species, strains, mutants andvariants of the genus Acetobacter or the genus Gluconacetobacter.

Specifically, DNA encoding a protein substantially identical to theprotein can be obtained from acetic acid bacteria belonging to the genusAcetobacter or the genus Gluconacetobacter, acetic acid bacteriabelonging to the genus Acetobacter or the genus Gluconacetobactertreated for mutagenesis, or natural mutants or variants thereof, by forinstance, isolating DNA: hybridizing under a stringent condition withDNA comprising a nucleotide sequence consisting of nucleotides 187 to1386 shown in SEQ. ID No. 1 in the sequence listing within thenucleotide sequence or with DNA comprising a nucleotide sequenceconsisting of nucleotides 110 to 1321 shown in SEQ. ID No. 3 in thesequence listing within the nucleotide sequence, and DNA: encodingprotein having a function of enhancing acetic acid tolerance. The termstringent condition here is a condition in which so-called specifichybrid is formed while non-specific hybrid is not formed. Though it isdifficult to quantify this condition clearly, if one example is taken, acondition in which DNA having high homology, for instance DNA havinghomology of 70% or more hybridizes, while DNA having homology lower thanthat does not hybridize, or a condition in which general washing isperformed for hybridization, for instance the washing is performed with0.1% SDS at the salt concentration equivalent to 1×SSC at 60° C., can beexemplified.

(2) The Acetic Acid Bacteria of the Present Invention

The acetic acid bacteria of the present invention mean bacteriabelonging to the genus Acetobacter or the genus Gluconacetobacter, orsuch bacteria, belonging to the genus Acetobacter or the genusGluconacetobacter which are enhanced their acetic acid tolerance.

In bacteria belonging to the genus Acetobacter, Acetobacter aceti can becited specifically, and Acetobacter aceti No. 1023 strain (deposited asFERM BP-2287 with International Patent Organism Depositary) can beexemplified.

Further, as for bacteria belonging to the genus Gluconacetobacter,Gluconacetobacter entanii can be cited, and Acetobacter altoacetigenesMH-24 strain currently deposited as FERM BP-491 with InternationalPatent Organism Depositary can be exemplified.

Acetic acid tolerance can be enhanced, for instance, by amplifyingintracellular copy numbers of acetic acid resistance-genes, ortransforming bacteria belonging to the genus Acetobacter by usingrecombinant DNA obtained by ligated DNA fragments containing structuralgenes to promoter sequences that function efficiently in the bacteriabelonging to the genus Acetobacter.

In addition, acetic acid tolerance can also be enhanced by replacing thepromoter sequence of the genes on chromosomal DNA with other promotersequence derived from microorganisms functioning efficiently in bacteriabelonging to the genus Acetobacter or the genus Gluconacetobacter, forinstance such promoters of other than acetic acid bacteria includingampicillin-resistance gene for plasmid pBR322, kanamycin-resistance genefor plasmid pACYC177, and chloramphenicol-resistance gene for plasmidpACYC184, B-galactosidase gene and the like from Escherichia coli.

Amplification of intracellular copy numbers of the genes can beconducted by introducing multi-copy vectors retaining the genes intocells of bacteria belonging to the genus Acetobacter, i.e. it can beconducted by introducing plasmid, transposon and the like retaining thegenes into cells of bacteria belonging to the genus Acetobacter or thegenus Gluconacetobacter.

As for multi-copy vectors, pMV24 (see non-patent literature 3, forinstance), pTA5001 (A), pTA5001 (B) (see patent literature 4, forinstance) and the like can be exemplified, and pMVL1 which is achromosome-integrative type vector (see non-patent literature 4, forinstance) can also be cited. Further, as for transposons, Mu, IS1452 andthe like can be exemplified.

Transformation of DNA into acetic acid bacteria belonging to the genusAcetobacter or the genus Gluconacetobacter can be performed by calciumchloride method (see non-patent literature 5, for instance),electroporation method (see non-patent literature 6, for instance) andthe like.

Enhancement of acetic acid tolerance in acetic acid bacteria belongingto the genus Acetobacter or the genus Gluconacetobacter having alcoholoxidation ability in the above-described manner, enables increase of theamount of production and production efficiency of acetic acid.

(3) Method of Producing Vinegar

Bacteria belonging to the genus Acetobacter or the genusGluconacetobacter, which are selectively enhanced their acetic acidtolerance by amplification of copy numbers of acetic acidresistance-genes in the above-described manner, and which have alcoholoxidation ability, are cultured on media containing alcohol, and aceticacid is produced and accumulated in the media. Vinegar can be thusefficiently produced.

The acetic acid fermentation in the method of producing of the presentinvention may be performed in the same manner as the conventional methodof producing vinegar by a method of fermentation by acetic acidbacteria. As for the medium used in acetic acid fermentation, it may beeither synthetic medium or natural medium as long as they are containingcarbon source, nitrogen source, inorganic substance and ethanol, andcontaining suitable amount of source of nutrition required by the usedbacteria strain for its growth if it is needed.

As for carbon sources, various kinds of carbohydrates including glucose,sucrose and various kinds of organic acids, can be exemplified. As fornitrogen source, natural nitrogen source such as peptone, degradationproduct of the microorganisms and the like can be exemplified.

In addition, static culture, shaking culture, aeration-agitation cultureand the like under aerobic condition are carried out, generally at theculture temperature of 30° C. The pH of medium is generally within therange of 2.5-7, preferably within the range of 2.7-6.5, and it also canbe adjusted with various kinds of acids, various kinds of bases, buffersand the like. Generally, after cultivation of 1 to 21 days acetic acidis accumulated at a high concentration in the medium.

(4) Embodiment of the Present Invention

Further, as recombinant plasmids pUSPT and pUSPT2 which are made byinserting ORF relating to the present invention or acetic acidresistance-gene comprising thereof (SEQ. ID No. 1 or SEQ. ID No. 3) intoEscherichia coli vector (multi-copy vector) pUC19, i.e. pUSPT has beendeposited as FERM BP-7932 with International Patent Organism Depositary,National Institute of Advanced Industrial Science and Technology,Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan since Mar. 1,2002, and pUSPT2 has been deposited as FERM BP-8304 with InternationalPatent Organism Depositary, National Institute of Advanced IndustrialScience and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba,Ibaraki, Japan since Feb. 26, 2003, so that DNA relating to the presentinvention can be available without difficulty, and a person skilled inthe art would easily carry out the present invention. In addition, if itis desired, by using these recombinant plasmids, ORF relating to thepresent invention or acetic acid resistance-genes comprising thereof,are transferred to the vectors capable of replicating autonomously inacetic acid bacteria, and these are introduced into acetic acid bacteriaand cultivated. By this procedure, vinegar having high acetic acidcontent, can be easily produced.

Still further, as explained above and as it is also apparent fromafter-mentioned examples, deposit of source of acetic acidresistance-genes, embodiment of PCR, preparation of plasmid vectors andrecombinant plasmids, deposit of host bacteria and the like, have beenelucidated, and each of them can be obtained, operated and processedeasily, therefore, performance of each operation and treatment accordingto Examples leads to obtainment of the objected acetic acidtolerance-transformants. The use of them enables to produce acetic acidat a high concentration. Consequently, the present invention can beeasily carried out in this point of view, as well.

The present invention will be explained more specifically with examplesbelow.

EXAMPLE Example 1 Cloning of Acetic Acid Resistance-Genes fromGluconacetobacter entanii and Determination of the Nucleotide Sequenceand the Deduced Amino Acid Sequence

(1) Generation of Chromosomal DNA Library

Acetbacter altoacetigenes MH-24 strain (FERMBP-491), that is one strainof Gluconacetobacter entanii was performed shaking culture at 30° C. onYPG medium (3% glucose, 0.5% yeast extract, and 0.2% polypeptone) added6% acetic acid and 4% ethanol. After the cultivation, culture medium wascentrifuged (7,500×g, 10 min) and bacterial cells were obtained.Chromosomal DNA was prepared from the obtained bacterial cells by themethod disclosed in patent literature 3.

The obtained chromosomal DNA as explained above was partially digestedby restriction enzyme PstI (Takara Shuzo Co., LTD.), further theEscherichia coli-acetic acid bacteria shuttle vector pMV24 wascompletely digested by restriction enzyme PstI. These types of DNA weremixed by adequate dose, ligated by using ligation kit (TaKaRa DNALigation Kit Ver. 2, Takara Shuzo Co., LTD.), and chromosomal DNAlibrary of Gluconacetobacter entanii was constructed.

(2) Cloning of Acetic Acid Resistance-Genes

The chromosomal DNA library of Gluconacetobacter entanii obtained asexplained above, was transformed into Acetobacter aceti No. 1023 strain(FERM BP-2287) that generally can grow only under up to approximatelyunder 1% acetic acid concentration on agar medium.

Subsequently, transformed Acetobacter aceti No. 1023 strain was culturedfor 4 days at 30° C. on YPG agar medium containing 2% acetic acid and100 μg/ml of ampicillin.

When colonies generated on the agar medium were inoculated into YPGmedium containing 100 μg/ml of ampicillin and cultured, and plasmidswere recovered from the obtained bacterial cells, the plasmid in whichPstI fragment of approximately 4 kbp was inserted, could be recovered asshown in FIG. 1 and this plasmid was named pP1. Further, it wasconfirmed that DNA fragment which enable Acetobacter aceti No. 1023strain to grow on YPG agar medium containing 2% acetic acid, wasapproximately 2 kbp of EcoRV-BalI fragment in approximately 4 kbp ofPstI fragment cloned into pP1.

The acetic acid resistance-gene fragment that enable to grow Acetobacteraceti No. 1023 strain on the agar medium containing 2% acetic acid,which strain usually can only be grown on agar medium containing up toapproximately 1% acetic acid concentration, was thus obtained.

(3) Determination of the Nucleotide Sequence of Cloned DNA Fragment

The cloned EcoRV-BalI fragment mentioned above was inserted into SmaIsite of pUC19, and the nucleotide sequence of the fragment wasdetermined by Sanger's dideoxy chain-termination method, and thenucleotide sequence described in SEQ. ID No. 1 was determinedconsequently. Sequencing was conducted in the all domains of the bothstrands of DNA, and it was also conducted so that cleavage sites wereoverlapped.

The presence of open reading frame (ORF) encoding 400 of amino acids(FIG. 3), as shown in SEQ. ID No. 2, was confirmed in nucleotides 187 to1386 of SEQ. ID No. 1.

Example 2 Enhancement of Acetic Acid Tolerance in Transformant,Transformed with Acetic Acid Resistance-Gene Derived fromGluconacetobacter entanii

(1) Transformation of Acetobacter aceti

Acetic acid resistance-gene derived from Acetobacter altoacetigenesMH-24 strain (FERM BP-491) cloned as explained above, was amplified byPCR method with KOD-Plus—(TOYOBO Co., LTD.), and plasmid pSPT in whichthe amplified DNA fragment was inserted into restriction enzyme SmaIcleavage site of the acetic acid bacteria-Escherichia coli shuttlevector pMV24 (see non-patent literature 3, for instance), wasconstructed. The outline of amplified fragment inserted into pSPT isshown in FIG. 1.

PCR method was performed as follows; i.e. PCR method was performed usinggenomic DNA derived from above-mentioned acetic acid bacterium astemplate and using primer 1 (the nucleotide sequence thereof is shown inSEQ. ID No. 5 (FIG. 7)) and primer 2 (the nucleotide sequence thereof isshown in SEQ. ID No. 6 (FIG. 8)) as primer in the following condition.

PCR method was performed i.e. for 30 cycles, in which 1 cycle isconsisting of performance at 94° C. for 15 sec, 60° C. for 30 sec, and68° C. for 2 min.

This pSPT was transformed into Acetobacter aceti No. 1023 strain byelectoroporation method (see non-patent literature 6, for instance). Thetransformants were selected on YPG agar medium added 100 μg/ml ofampicillin and 2% acetic acid.

Plasmid was extracted from the transformant having ampicillin resistancegrown on the selection medium in the conventional manner for analysisand it was confirmed that it retained the plasmid having acetic acidresistance-gene.

(2) Acetic Acid Tolerance of the Transformant

The ampicillin-resistant transformant having plasmid pSPT obtained asexplained above was compared their growth on YPG medium added aceticacid to that of the original strain of Acetobacter aceti No. 1023 strainintroduced only shuttle vector pMV24.

Specifically, the transformant including pSPT and the original strainhaving shuttle vector pMV24 were inoculated into 100 ml of YPG mediumcontaining 3% ethanol and 100 μg/ml of ampicillin and into 100 ml of YPGmedium containing 3% ethanol, 3% acetic acid and 100 μg/ml ofampicillin, respectively, and shaking culture (150 rpm) was performed at30° C., and growth of the transformant in the medium containing aceticacid was compared to that of the original strain by measuring absorbanceat 660 nm.

Consequently, it was confirmed that the transformant and the originalstrain Acetobacter aceti No. 1023 could grow almost similarly in themedium without containing acetic acid, while the transformant could growbut the original strain could not grow in the medium containing 3%acetic acid and 3% ethanol, as shown in FIG. 2. Function of enhancingacetic acid tolerance of acetic acid resistance-gene was confirmed.

(3) Thermal Resistance of the Transformant

The ampicillin-resistant transformant having plasmid pSPT obtained inabove-mentioned (1), was compared their growth on YPG medium in whichthe cultivation temperature was changed, to that of the original strainof Acetobacter aceti No. 1023 introduced only shuttle vector pMV24.

Specifically, they were cultured with agitation rate at 400 rpm,aeration rate at 0.2 vvm and temperature of 30° C. in 1 L of YPG mediumcontaining 1% acetic acid, 4% ethanol, and 100 μg/ml of ampicillin using2 L of mini-jar fermenter (Chiyoda Seisakusho Co., LTD.: TBR-2-1), andfermented up to approximately 3% acetic acid concentration. Then, theculture medium was withdrawn from the mini-jar fermenter with 200 ml ofit left, 800 ml of YPG medium containing acetic acid, ethanol and 100μg/ml of ampicillin was newly added, it was adjusted to theconcentration of 4% ethanol and 1% acetic acid, culture temperature wasrisen to 33° C., and fermentation was thus restarted.

When fermentation further progressed and concentration of acetic acid inthe medium became approximately 3%, the culture medium was withdrawnagain and the medium was added again, further culture temperature wasrisen to 36° C. to ferment them similarly, and still further acetic acidfermentation was performed by raising temperature 1° C. at a time in thesame manner.

Then, growth of bacteria was compared by measuring absorbance at 660 nm,and the ratio of acetic acid fermentation was compared by measuringacetic acid concentration in culture medium.

As a result, acetic acid fermentation and growth of bacteria at 40° C.were possible in the transformant, while acetic acid fermentation andgrowth of bacteria could confirmed only up to 37° C. in the originalstrain of Acetobacter aceti No. 1023 as shown in FIG. 3, and thefunction of enhancing acetic acid tolerance of SPT gene was confirmed.

Example 3 Acetic Acid Fermentation of the Transformant Transformed withAcetic Acid Resistance-Gene Derived from Gluconacetobacter entanii

The ampicillin-resistant transformant having plasmid pSPT obtained inExample 2 was compared with fermentation ability to that of the originalstrain of Acetobacter aceti No. 1023 having only shuttle vector pMV24.

Specifically, they were cultured with agitation rate at 400 rpm,aeration rate at 0.20 vvm and temperature of 30° C. in 2.5 L of YPGmedium containing 1% acetic acid, 4% ethanol and 100 μg/ml of ampicillinusing 5L of mini-jar fermenter (Mitsuwa Rikagaku Kogyo Co.; KMJ-5A), andfermented up to 3% acetic acid concentration. Then, the culture mediumwas withdrawn from the mini-jar fermenter with 700 ml of it left, 1.8 Lof YPG medium containing 100 μg/ml of ampicillin was newly added, it wasadjusted to the concentration of 4% ethanol and 1% acetic acid, andfermentation was thus restarted. The concentration of ethanol in themedium was maintained at 1% by addition of ethanol during thefermentation, and still further acetic acid fermentation was performed.Then, acetic acid fermentation ability of the transformant was comparedto that of the original strain. The result was summarized in Table 1.TABLE 1 Final acetic acid Specific Production Growth concentra- growthrate rate lag-time tion (%) (OD660/hr) (%/hr) (hr) Original 9.5 0.01510.103 62.5 strain Transformant 11.1 0.0323 0.136 24.0

From the result of Table 1, it was confirmed that the transformant wasapparently superior in any of final acetic acid concentration, specificgrowth rate, production rate, and growth lag-time.

Example 4 Enhancement of Acetic Acid Tolerance in the TransformantTransformed with Acetic Acid Resistance-Gene Derived fromGluconacetobacter entanii

(1) Transformation of Acetobacter altoacetigenes

Plasmid pSPT obtained in Example 2 was transformed into Acetobacteraltoacetigenes MH-24 strain (FERMBP-491) which was one strain ofGluconacetobacter entanii by electroporation method (see non-patentliterature 6, for instance). The transformant was selected on YPG agarmedium containing 0.55% agar added 100 μg/ml of ampcillin, 4% aceticacid and 4% ethanol.

The ampicillin-resistant transformant grown on the selection medium wasextracted plasmid in the conventional manner for analysis and it wasconfirmed that they retained the plasmid having SPT gene.

Specifically, they were cultured with agitation rate at 500 rpm,aeration rate at 0.20 vvm and temperature of 30° C. in 2.5 L of YPGmedium containing 4% acetic acid, 4% ethanol, and 100 μg/ml ofampicillin using 5 L of mini-jar fermenter (Mitsuwa Rikagaku Kogyo Co.;KMJ-5A), and fermented up to 6.3% acetic acid concentration. Then, theculture medium was withdrawn from the mini-jar fermenter with 700 ml ofit left, 1.8 L of YPG medium containing 100 μg/ml of ampicillin wasnewly added, it was adjusted to the concentration of 6% and 4% ethanol,and fermentation was thus restarted. The concentration of ethanol in themedium was maintained at 1% by addition of ethanol during thefermentation, and still further acetic acid fermentation was performed.Then acetic acid fermentation ability of the transformant was comparedto that of the original strain. The result was summarized in Table 2.TABLE 2 Final acetic acid Specific Production concentration growth raterate (%) (OD660/hr) (%/hr) Original 14.6 0.501 0.142 strain Transformant16.2 0.756 0.175

From the result of Table 2, it was confirmed that transformants wereapparently superior in any of final acetic acid concentration, specificgrowth rate, production rate, and growth lag-time.

Example 5 Cloning of Acetic Acid Resistance-Gene from Acetobacter acetiand Determination of the Nucleotide Sequence and the Deduced Amino AcidSequence

Acetobactger aceti No. 1023 strain (FERM BP-2287) was performed shakingculture on YPG medium (3% glucose, 0.5% yeast extract and 0.2%polypeptone) at 30° C. for 24 h. After the cultivation, culture mediumwas centrifuged (7,500×g, 10 min) and bacterial cells were obtained.Chromosomal DNA was prepared from obtained bacterial cells bychromosomal DNA preparation method (see patent literature 3, forinstance).

Cloning was conducted by inverse PCR method using above-prepared DNA asa template. Specifically, primer 3 (the nucleotide sequence thereof isshown in SEQ. ID No. 7 (FIG. 9)) and primer 4 (the nucleotide sequencethereof is shown in SEQ. ID No. 8 (FIG. 10)) were synthesized fromdomains which were thought to have higher conservation in comparisonwith other species from DNA sequence (SEQ. ID No. 1) obtained inAcetobacter altoacetigenes MH-24 strain. Next, PCR reaction was carriedout using chromosomal DNA of Acetobacter aceti No. 1023 strain as atemplate, and approximately 750 bp of amplified fragments were obtained.Then, chromosomal DNA of Acetobacter aceti No. 1023 strain wascompletely digested by restriction enzyme PstI, and legations wereperformed in the conventional manner. PCR was performed using primer 5(the nucleotide sequence thereof is shown in SEQ. ID No. 9 (FIG. 11))and primer 6 (the nucleotide sequence thereof is shown in SEQ. ID No. 10(FIG. 12)) and using the ligation product as a template andapproximately 3 kbp of amplified fragment was obtained.

As a result of sequencing the nucleotide sequences of this fragment bySanger's dideoxy chain-termination method using above-mentioned primers,the nucleotide sequence shown in SEQ. ID No. 3 was determined.Sequencing was conducted in the all domains of the both of strands ofDNA.

Example 6 Enhancement of Acetic Acid Tolerance in the TransformantTransformed with Acetic Acid Resistance-Gene Derived from Acetobacteraceti

(1) Transformation of Acetobacter altoacetigenes

Plasmid pSPT2 obtained in Example 5 were transformed into Acetobacteraltoacetigenes MH-24 strain (FERM BP-491) which was one strain ofGluconacetobacter entanii by electroporation method (see non-patentliterature 6, for instance). The transformants were selected on YPG agarmedia containing 0.55% agar added 100 μg/ml of ampcillin, 4% acetic acidand 4% ethanol.

The transformants having ampicillin tolerance, grown on the selectionmedium, were extracted plasmids in the conventional manner for analysisand it was confirmed that they retained the plasmids including SPT gene.

Specifically, they were cultured with agitation rate at 500 rpm,aeration rate at 0.20 vvm and temperature of 30° C. in 2.5 L of YPGmedium containing 4% acetic acid, 4% ethanol, and 100 μg/ml ofampicillin using 5 L of mini-jar fermenter (Mitsuwa Rikagaku Kogyo Co.;KMJ-5A), and fermented up to 6.3% acetic acid concentration. Then, theculture medium was withdrawn from the mini jar fermenter with 700 ml ofit left, 1.8 L of YPG medium containing 100 μg/ml of ampicillin wasnewly added, it was adjusted to the concentration of 6% and 4% ethanol,and fermentation was thus restarted. The concentration of ethanol in themedium was maintained at 1% by addition of ethanol during thefermentation, and still further acetic acid fermentation was performed.Then, acetic acid fermentation ability of the transformant was comparedto that of the original strain. The result was summarized in Table 3.TABLE 3 Final acetic acid Specific Production concentration growth raterate (%) (OD660/hr) (%/hr) Original 14.6 0.501 0.142 strain Transformant16.0 0.605 0.153

From the result of Table 3, it was confirmed that the transformants wereapparently superior in any of final acetic acid concentration, specificgrowth rate, production rate, and growth lag-time.

INDUSTRIAL APPLICABILITY

According to the present invention, novel genes participating in aceticacid tolerance can be provided, further bred strain capable ofefficiently producing vinegar at higher acetic acid concentration byusing the genes can be obtained. Still further, the present inventionenables to furnish a method of efficiently producing vinegar at higheracetic acid concentration using the bred strain.

1. A protein SPT described in following (A) or (B): (A) A protein havingan amino acid sequence shown in SEQ. ID No. 2 in the sequence listing.(B) A protein consisting of an amino acid sequence comprisingsubstitution, deletion, insertion, addition, or inversion of one orseveral amino acids in an amino acid sequence shown in SEQ. ID No. 2 inthe sequence listing and having a function of enhancing acetic acidtolerance.
 2. A DNA of a gene encoding the protein SPT described infollowing (A) or (B): (A) A protein having an amino acid sequence shownin SEQ. ID No. 2 in the sequence listing. (B) A protein consisting of anamino acid sequence comprising substitution, deletion, insertion,addition, or inversion of one or several amino acids in an amino acidsequence shown in SEQ. ID No. 2 in the sequence listing and having afunction of enhancing acetic acid tolerance.
 3. The DNA of a geneaccording to claim 2, that is a DNA described in following (a) or (b):(a) A DNA that comprises a nucleotide sequence consisting of nucleotides187 to 1386 shown in SEQ. ID No. 1 in the sequence listing within thenucleotide sequence. (b) A DNA that hybridizes with a probe comprising anucleotide sequence consisting of nucleotides 187 to 1386 shown in SEQ.ID No. 1 in the sequence listing within the nucleotide sequence or apart thereof under a stringent condition, and encodes protein having afunction of enhancing acetic acid tolerance.
 4. A protein SPT2 describedin following (A) or (B): (A) A protein having an amino acid sequenceshown in SEQ. ID No. 4 in the sequence listing. (B) The protein SPT2consisting of an amino acid sequence comprising substitution, deletion,insertion, addition, or inversion of one or several amino acids in anamino acid sequence shown in SEQ. ID No. 4 in the sequence listing andhaving a function of enhancing acetic acid tolerance.
 5. A DNA of a geneencoding the protein SPT2 described in following (A) or (B): (A) Aprotein having an amino acid sequence shown in SEQ. ID No. 4 in thesequence listing. (B) The protein SPT2 consisting of an amino acidsequence comprising substitution, deletion, insertion, addition, orinversion of one or several amino acids in an amino acid sequence shownin SEQ. ID No. 4 in the sequence listing and having a function ofenhancing acetic acid tolerance.
 6. The DNA of a gene according to claim5, that is a DNA described in following (A) or (B): (A) A DNA thatcomprises a nucleotide sequence consisting of nucleotides 386 to 1636shown in SEQ. ID No. 3 in the sequence listing within the nucleotidesequence. (B) A DNA that hybridizes with a probe generated from anucleotide sequence consisting of nucleotides 386 to 1636 shown in SEQ.ID No. 3 in the sequence listing within the nucleotide sequence or apart thereof under a stringent condition, and encodes protein having afunction of promoting growth rate.
 7. Microorganisms, wherein aceticacid tolerance thereof is enhanced by amplifying intracellular copynumber of the DNA according to any one of claims 2, 3, 5, or
 6. 8. Themicroorganisms according to claim 7, wherein the microorganisms areacetic acid bacteria belonging to the genus Acetobacter or the genusGluconacetobacter.
 9. A method of producing vinegar, whereinmicroorganisms having alcohol oxidation ability among the microorganismsaccording to claim 7 or 8, are cultured on medium containing alcohol sothat acetic acid is produced and accumulated in the medium.
 10. Arecombinant plasmid pUSPT (FERM BP-7932) including the DNA according toclaim 2 or 3, or a recombinant plasmid pUSPT2 (FERM BP-8304) includingthe DNA according to claim 5 or 6.